Mechanism Analysis Of An RNA Silencing Suppressor NgRBP Encoded By Nicotiana Glutinosa Involving In Hypersensitive Response

RNA silencing is a kind of sequence-specific degradation mechanism commonly found in eukaryotic organisms to involve in resisting the invasion of viruses and heterologous nucleic acid.To counteract host defense,many plant viruses encode viral suppressors of RNA silencing?VSRs?to guarantee their successful invasion.Plants also encode endogenous suppressors of RNA silencing?ESRs?to participate in appropriate regulation of RNA silencing.But the mechanism of ESRs to host defense is meaningful but unclear.Thus,the study on the ESRs will contribute to further explore the regulatory mechanism of RNA silencing and the complex interactions between viruses and host plants.Nicotiana glutinosa is a subgroup of tobacco containing N gene.N.glutinosa triggers a strong hypersensitivity response to form necrotic spots infected by Tobacco mosaic virus?TMV?,and also significantly up-regulates the expression of an ESR,NgRBP,suggesting that NgRBP may be involved in the resistance to TMV of N.glutinosa.In this study,we observed the response to TMV by up-regulating and down-regulating NgRBP.NgRBP was able to accelerate and enhance HR induced by TMV.HR quickly inducted a variety of intracellular physiological and biochemical processes.So we explored the molecular mechanism of HR enhancement by intensive time interval sampling.The main research results are as follows:?1?NgRBP was able to inhibit RNA silencing and affect the expression of key genes in silencing pathways.At 5dpi,the green fluorescence intensity was strong and the GFP siRNAs level was drastically undetected in N.benthamiana line 16c leaves coinfiltrated with GFP and NgRBP.It was demonstrated that NgRBP was able to inhibit RNA silencing.At3dpi,the expression of silencing-related genes was detected,which showed that the expression of DCL1 and AGO6 were about 40%of empty vector control.At 3dpi,GFP mRNA accumulation was less than 35S-GFP expression vector in N.benthamiana leaves coinfiltrated with DCL1 promoter fusion GFP expression vector and NgRBP.Chromosome immunoprecipitation?ChIP?analysis of leaves infiltrated with NgRBP fusion GFP expression vector at 3dpi,showed that NgRBP did not bind to DCL1 promoter.NgRBP down-regulates the key genes in silencing pathways to suppress RNA silencing.NgRBP may affect the expression of DCL1 through other transcription factors.?2?NgRBP was able to enhance HR and systemic acquire resistances induced by TMV.HR induced by TMV in N.glutinosa,formed necrotic spots,and induced NgRBP up-regulation.It was found that the number and the size of necrotic spots were decreased and the browning of the edge was increased in N.glutinosa leaves inoculated with TMV after overexpression of NgRBP,compared with empty vector control,which indicated that the death cells were decreased.H₂O₂ content increased by Trypan blue and 3,3?-diaminobenzidie?DAB?staining by overexpressing NgRBP.The results of qRT-PCR showed that the accumulation of TMV was decreased and the expression of pathogenesis-related protein genes PR1,PR2 and PR5 were significantly increased.It is suggested that NgRBP may enhance the HR induced by TMV.TMV secondary inoculation exhibited that the phenomenon of the upper leaves was consistent with the local inoculation.HR and SAR in NgRBP down-regulation plants were weaker than normal N.glutinosa and NgRBP transient overexpressing plants.These results showed that NgRBP not only enhanced the local HR induced by TMV,but also enhanced SAR.?3?Overexpression of NgRBP was able to accelerate the occurrence of HR.At 1dpi,empty vector and NgRBP were infiltrated in N.glutinosa leaves respectively,then the infiltrated leaves were inoculated TMV.At 36h postinoculation?hpi?with TMV,NgRBP infiltrated leaves were observed visible necrotic spots 8h earlier than that of empty vector and 12h earlier than TMV.Analysis of TMV accumulation indicated that the accumulation of TMV in NgRBP infiltrated leaves was faster and earlier than that of empty vector,and it reached maximal level in advance,while the low level of TMV accumulation in NgRBP infiltrated leaves might be due to the induction of a stronger HR.The expression of HR maker genes hin1 and hsp203 were induced in advance.At the same time,the expression levels of ROS synthesis-related genes RbohA,RbohB and SA synthesis-related gene pal were significantly increased and peaked in advance.The expression level of ROS-scavenging gene POD was significantly decreased.These results further suggested that NgRBP does facilitate the occurrence of HR in advance.TMV accumulated lagging and slow when the expression of NgRBP was down-regulated by VIGS.Due to a weak HR,the overall amount of virus accumulation was higher,the expression of these genes were lag behind compared with NgRBP overexpression lines.These results suggested that NgRBP promoted the accumulation of TMV in the early stage of TMV infection by the silencing suppression function of NgRBP,accelerated the recognition of N gene and TMV,and leaded to the occurrence of HR in advance.The accumulation of ROS and SA were increased,the replication of TMV amplification were limited,reducing the number of necrotic spots and dead cells.?4?NgRBP enhanced local and systemic RNA silencing induced by HR.Studies have shown that HR was able to induce local and systemic RNA silencing,the leaves coinfiltrated with NgRBP and GFP were inoculated TMV,GFP mRNA accumulation was significantly reduced and siRNA accumulation was increased compared with infiltrated with GFP alone.The above results indicated that NgRBP enhanced local RNA silencing by enhancing HR.The leaves after overexpressing and down-regulating NgRBP were inoculated TMV respectively,at the same time,then the upper leaves were infiltrated with GFP.It was found that NgRBP could enhance systemic RNA silencing induced by HR.It is speculated that NgRBP enhanced systemic RNA silencing by promoting HR.So SAR induced by HR especially for viruses may be partially achieved by enhancing RNA silencing.?5?The enhancement of HR by NgRBP may be only related to its silencing suppression function.Two kinds of RNA silencing suppressor were used to construct plant expression vector,P19 encoded by Tomato bushy stunt virus?TBSV?and HVT063 encoded by turkey spider virus?HVT?.The number and the size of necrotic spots were reduced,TMV accumulation was significantly reduced in N.glutinosa leaves overexpressing P19 and HVT063,which was consistent with those of NgRBP.The results of qRT-PCR showed that at12hpi with TMV,the expression of hin1 was significantly higher than that of empty vector control.These results suggested that HVT063 and P19 were able to enhance HR.Therefore,it is speculated that the enhancement of HR is likely to be achieved by suppressing RNA silencing,not the specific function of NgRBP.